Fibrous protein-immobilization systems

ABSTRACT

The present invention provides a fibrous protein-immobilization system composition comprising a fiber comprising fiber-forming material, and a protein attached to the fiber-forming material.

This application is a 371 of PCT/US03/19197, filed Jun. 18, 2003, whichclaims the benefit of U.S. Provisional Application 60/389,537 filed Jun.18, 2002, which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to fibrous protein-immobilization systems andmethods for their synthesis and use.

BACKGROUND OF THE INVENTION

Enzymatic biotransformations have been pursued extensively for manyimportant chemical processing applications such as chemical production,drug synthesis, pollutant degradation, and petroleum refining because oftheir unparalleled selectively and mild reaction conditions. In manycases, however, low catalytic efficiency and stability of enzymes havebeen seen as barriers for the development of large-scale operations tocompete with traditional chemical processes.

In practice, almost all large-scale industrial operations preferablyemploy immobilized enzymes because they afford easy recycling, feasiblecontinuous operations, and simplified product purification. Many methodshave been developed to incorporate enzymes into a variety of organic andinorganic solid supports, entrap enzymes in hollow fibers ormicrocapsules, and cross-link enzymes via covalent bonds. Among otherfactors, the structure of the support materials has a great impact onthe performance of the immobilized enzymes. Nonporous support materials,to which enzymes are attached at the surfaces, are subject to minimumdiffusional limitations. However, enzyme loading per unit mass ofsupport is usually low. Alternatively, high enzyme loading can beachieved with porous materials such as membranes, gel matrices, andporous materials. Porous materials, however, suffer much greaterdiffusional limitation. For example, the value of effectiveness factor(η, which measures the ratio of apparent heterogeneous reaction rate tohomogeneous reaction rate) for α-chymotrypsin entrapped inpolyacrylamide hydrogel was reported to be ˜0.3; for α-chymotrypsinincorporated into hydrophobic plastics, η was below 0.1; forcross-linked α-chymotrypsin, η was below 10⁻³. Higher η values arepossible for the same immobilized enzyme but used for nonaqueousreactions, mostly due to the relatively slower reaction rates involvedthere.

The reduction in size of support materials can effectively improve theefficiency of immobilized enzymes. In some cases, such at in the case ofsurface attachment on non-porous materials, smaller particles have beenshown to provide relatively higher enzyme loading per unit mass. Forporous materials, smaller particles are subject to much reduceddiffusional resistance because of a shortened path of diffusion.

Many studies on the use of micrometer sized materials have beenconducted. However, it has only been recently that even smaller scalematerials have been studied. In these newer studies, nanoparticles havebeen used as carriers or supports for enzyme immobilization. Theeffective enzyme loading on nanoparticles can be very high, and a largesurface area per unit mass is also available to facilitate reactionkinetics. It will be appreciated that the enzymes are attached to thenanoparticles.

While the use of nanoparticles provide excellent results in terms ofbalancing the contradictory issues of surface area, diffusionresistance, and effective enzyme loading, their ability to be dispersedin reaction solutions and their subsequent recovery for reuse aredauntingly difficult.

There is, therefore, still a need for immobilizing enzymatic catalystsby employing substrates that have the benefits of nanosized materials,e.g., have relatively high enzyme loading capability, have large surfacearea, and have minimal diffusional limitations, but yet are easilyrecoverable for reuse or continuous use.

SUMMARY OF THE INVENTION

The present invention provides a fibrous protein-immobilization systemcomposition comprising a fiber comprising fiber-forming material, and aprotein attached to the fiber-forming material.

The present invention further provides a method for synthesizing afibrous protein immobilization system comprising the steps ofsynthesizing a fiber comprising a fiber-forming material, and attachinga protein to the fiber-forming material.

The present invention advantageously overcomes problems in the prior artby immobilizing proteins via attaching them to the fiber-formingmaterial of a fiber. Advantages provided by the fibrousprotein-immobilization system include: relatively high surface area perunit mass of the fibrous protein-immobilization system, relativelyreduced diffusional resistance, relatively high protein loading upon thefiber, and relative ease of recovery. Further, enzyme activity andstability is significantly enhanced with the fibrousprotein-immobilization systems.

Fibrous protein-immobilization systems can be employed in a variety ofapplications including:

-   -   A. Industrial bioprocessing for chemical production, drug        synthesis, pollutant degradation, fuel processing, and the like.    -   B. Constructing bioactive filtration materials that can be used        to prepare higher performance barriers, fabrics; constructing        filters that employ enzymes for degrading volatile organic        pollutants in order to improve air quality; and degrading        chemical warfare agents such as nerve gas.    -   C. Producing biosensors that can be used for clinical,        environmental, and military applications.    -   D. Constructing energy devices by employing redox enzymes into        electro-conductive fibers (such as those of polyaniline). The        fibers can then be used to construct bio-electrodes that        generate electrical power using biomaterials including sugar,        alcohol, and urea as energy sources.

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

Fibrous protein-immobilization systems (fibrous systems) compriseproteins that are attached to the fiber-forming materials of a fiber.The term “attached” refers to two substances being linked to each otherby a chemical bond, e.g., a protein attached to a fiber-formingmaterial. Fibrous systems are synthesized by attaching proteins tofiber-forming material before or after the fiber-forming material isprocessed into a fiber. Proteins can be attached to a fiber-formingmaterial directly or indirectly. Direct attachment occurs where achemical bond exists between a protein and a fiber-forming material, andindirect attachment occurs where an inert coupling agent links a proteinto a fiber-forming material.

Both natural and synthetic proteins can be employed in fibrous systems.Non-limiting examples of useful proteins include enzymes, hormones,toxins, antibodies, antigens, lectins, structural proteins, signalproteins, transport proteins, receptors, and blood factors. Proteinsgenerally include at least one functional group that can react with acorresponding functional group on a fiber-forming material or couplingagent and thereby create a point of attachment. For example, mostnaturally occurring proteins include at least one of the followingfunctionalities: amine [(RNH₂), R(NH), C(NH), and (NH₂)], sulfhydryl(RSH), carboxyl (RCOOH), and phenol (RC₆H₄OH). Enzymes are preferablyemployed in fibrous protein-immobilization systems, and examples ofpreferred enzymatic functional groups are:

Enzymes can catalyze chemical reactions. Some preferred enzymes employedin fibrous systems include those having biochemical activities such aschymotrypsin, cytochrome C, trypsin, subtilisin, horseradish peroxidase,soybean peroxidase, and glucose oxidase. Especially preferred enzymesare α-chymotrypsin, β-galactosidase, and chloroperoxidase.

Some enzymes catalyze reactions because of the amino acid residues thatform their polypeptide chains; an example is pancreatic ribonuclease.Other enzymes, however, require an additional chemical component calleda cofactor for catalytic activity. The cofactor can be an inorganiccompound or element, such as Fe²⁺, Mn²⁺, or Zn²⁺ ions, or it may be acomplex organic molecule called a coenzyme; coenzymes act as carriers ofspecific functional groups. Some enzymes require both a coenzyme and oneor more metal ions for activity. In some enzymes, the coenzyme or metalion is only loosely and transiently bound to the protein, but in othersit is tightly and permanently bound via covalent chemical bonds, inwhich case it is called a prosthetic group. A complete catalyticallyactive enzyme together with its coenzyme or metal ion is called aholoenzyme. The protein part of the holoenzyme is called the apoenzyme.

The protein loading of a fibrous protein-immobilization system can befrom about 0.01% to about 99% weight of the fibrous system. Preferably,protein loading is from about 0.1% to about 10% weight of the fibroussystem, and more preferably protein loading is from about 1% to about10% weight of the fibrous system.

Fibers employed in the present invention can comprise a variety offiber-forming materials, which includes any polymer that can bedissolved in a solvent. Preferably, a polymer that retains itsmechanical strength while swollen with solvents, reactants, or reactionproducts is employed as a fiber-forming material because of itsdurability under conventional chemical process operating conditions.More preferably, polymeric fiber-forming materials are employed insynthesizing fibers that can be crosslinked into a strong network.

Fiber-forming material can comprise chemical functionalities that allowthe fiber-forming material to attach to either a protein or couplingagent. For instance, a fiber-forming material could be a functionalizedpolymer that derives from either a natural or synthetic polymer.Examples of useful fiber-forming materials include, but are not limitedto, polymers such a nylon, polyacrylonitrile (PAN), polyesters,polyurethanes, silanes, or copolymers thereof. Useful synthetic polymersthat can be employed as fiber forming materials are: plastics, solidpowders, resins, and waxes that are linear and soluble in a solvent suchas chloroform, dimethylformide, or toluene. A polymeric fiber-formingmaterial that is preferably employed is polystyrene. Polymericfiber-forming materials can be synthesized or modified usingconventional techniques so that they comprise an appropriate functionalgroup that will allow them to attach to a protein or coupling agent.

Any functional group that will allow a fiber-forming material to attachto a coupling agent or protein can be employed in the fibrous system.Non-limiting examples of useful functional groups on fiber-formingmaterials include hydroxy groups (—OH), amine groups (RNH₂ and (R₂NH),sulfhydryl groups (RSH), carboxyl groups (—COOH), and aldehyde groups[—C(O)H].

Non-limiting examples of preferred functional groups for fiber-formingmaterials are:

Linear polymers are examples of fiber-forming materials that can bemodified to include a functional group. Linear polymers include but arenot limited to: homopolymers and copolymers of α-olefins,α-β-ethylenically unsaturated carboxylic acids, vinyl aromatics, ethylethers, and combinations thereof. Exemplary α-olefins include: ethylene,propylene, pentene 1-butene, 1-hexene, 4methyl-1 pentene, 1-octene,1-decene, or combinations thereof. Exemplary α-β-ethylenicallyunsaturated carboxylic acids include: acrylic acid, methacrylic acid.Exemplary vinyl aromatics include styrene. Exemplary ethyl ethersinclude: ethylene glycol, vinyl ethyl ether, vinyl acetate, and methylmethacrylate. Preferred polymers are: polyethylene glycol, polystyrene,poly(methyl methacrylate), poly(vinyl acetate), and poly(vinyl ethylether).

Without undue experimentation, a person of ordinary skill in the art canselect and employ a fiber-forming material having a functional groupthat will react with a corresponding functionality on a protein orcoupling agent and thereby attach the two together.

The fiber-forming material loading of a fibrous system can be from about0.01% to about 99% weight of the fibrous system. Preferably,fiber-forming material loading is from about 0.1% to about 90% weight ofthe fibrous system, and more preferably protein loading is from about 1%to about 80% weight of the fibrous system.

Coupling agents can be employed in fibrous systems to serve as a linkbetween fiber-forming materials and proteins. Useful coupling agentsinclude compounds that are preferably inert except for having at leasttwo reactive functional groups wherein at least one of the couplingagent's functional groups can react with a functionality on a proteinand at least another of its functional groups can react with afunctionality on a fiber-forming material.

Non-limiting examples of preferred coupling agents are:

The loading of coupling agents that can be employed in a fibrousprotein-immobilization system can make up from about 0.0001% to about50% of the total weight of the fibrous system. Preferably, couplingagents make up from about 0.001% to about 1% of the total weight of thefibrous system, and more preferably coupling agents make up from about0.001% to about 0.1% of the total weight of the fibrous system.

Various conventional techniques that can be used to form fibers can beemployed in synthesizing fibrous catalyst-immobilization systems,however electrospinning is preferred. The technique of electrospinningof solutions containing fiber-forming material, is known and has beendescribed in a number of patents as well as in the general literature.Electrospinning involves introducing a solution into an electric field,whereby the solution is caused to produce fibers that tend to be drawnto an electrode. While being drawn from the solution, the fibers usuallyharden, which may involve mere cooling (e.g., where the liquid isnormally solid at room temperature), chemical hardening (e.g., bytreatment with a hardening vapor), or evaporation of solvent (e.g., bydehydration). The product fibers may be collected on a suitably locatedreceiver and subsequently stripped from it. Electrospinning can producefibers from a great variety of fiber-forming materials, and the fiberscan have diameters greater than or equal to about two nanometers.

When electrospinning is employed, any solvent in which a fiber-formingmaterial is soluble can be used to prepare solutions that can be used tosynthesize fibrous systems. Therefore, when preparing solutionscomprising fiber-forming materials, persons of ordinary skill in the artcan employ appropriate solvents based on the solubility characteristicsof fiber-forming material(s) without undue experimentation.

The percent concentration of a solvent in a solution for electrospinningcan be from about 0% to about 99% volume of the solution. Preferablysolvent is employed from about 0% to about 85% volume of the solution.More preferably, solvent is employed from about 0% to about 75% volumeof the solution.

The percent concentration of fiber-forming material in a solution forelectrospinning can be from about 0% to about 99% volume of thesolution. Preferably fiber-forming material is employed from about 0% toabout 85% volume of the solution. More preferably, fiber-formingmaterial is employed from about 0% to about 75% volume of the solution.

Proteins can be attached to fiber-forming materials directly orindirectly. Direct attachment occurs where a protein chemically bonds toa fiber-forming material, and indirect attachment occurs where acoupling agent links a protein to a fiber-forming material.

In order to directly attach a protein to a fiber-forming material, achemical bond must form between the two. Direct attachment is preferablyachieved by presenting a protein to a fiber-forming material via asolution comprising proteins. Fiber-forming materials are preferablyimmersed or dissolved into the solution for a sufficient time thatallows the chemical functionalities on the fiber-forming material tobond to the proteins. Where two immiscible solutions are employed, onecomprising proteins and the other comprising fiber-forming material,agitation or convention emulsion techniques are preferably employed.Additionally, proteins can also be presented to fiber-forming materialsvia mist or using other conventional methods.

Where proteins in solution are being directly or indirectly attached tofiber-forming material in solution and that has not yet been processedinto a fiber, the solution comprising proteins preferably has aconcentration of proteins ranging from about 0.001% to about 50% weightof the solution. Preferably, the concentration of proteins in solutionranges from about 0.01% to about 10% weight of solution. Morepreferably, the concentration of proteins in solution ranges from about0.01% to about 1% weight of solution.

When proteins are being directly attached to fiber-forming material thathas been processed into a fiber, the fiber is preferably immersed in asolution comprising proteins wherein the concentration of proteins inthe solution can be from about 0.001% to about 50% by weight of thesolution. Preferably the concentration of proteins in solution is fromabout 0.001% to about 10% weight of the solution, and more preferablythe concentration of proteins in solution is from about 0.01% to about1% weight of the solution.

Indirect attachment of a protein to a fiber-forming material requiresthat a coupling agent serve as a link between the two. The protein andfiber-forming material preferably possess chemical functionalities thatwill chemically bond to the coupling agent. And where a coupling agentis employed, the coupling agent is preferably first attached to thefiber-forming material and then the protein. The preferred sequence ofindirect attachment can be achieved by immersing or dissolving afiber-forming material into a solution comprising coupling agents for asufficient time that allows the fiber-forming materials to bond to thecoupling agents. Following this first attachment, the coupling agent(s)are then preferably introduced into a separate solution comprisingproteins for a sufficient time that allows the coupling agent'sfunctionalities to chemically bond to the proteins.

Other alternative methods for indirect attachment can include varioussequences of attachment involving solutions of fiber-forming material,coupling agents, proteins, and combinations thereof. Where two solutionsare immiscible, agitation and conventional emulsion techniques arepreferably employed.

Where coupling agents in solution are being attached to fiber-formingmaterial in solution and that has not been processed into a fiber, thesolution comprising coupling agents preferably has a concentration ofcoupling agents ranging from about 0.001% to about 50% weight of thesolution. Preferably, the concentration of coupling agents in solutionranges from about 0.01% to about 1% weight of solution. More preferably,the concentration of coupling agents in solution ranges from about 0.01%to about 0.1% weight of solution.

In order to attach coupling agents to fiber-forming materials that havebeen processed into a fiber, the fiber is preferably immersed in asolution comprising coupling agents wherein the concentration ofcoupling agents in solution ranges from about 0.001% to about 100% byweight of the solution. Preferably the coupling agents have aconcentration in the solution ranging from about 0.001% to about 1%weight of the solution, and more preferably the coupling agents have aconcentration ranging from about 0.01% to about 0.1% weight of thesolution.

Where a single solution comprises coupling agents and proteins in orderto attach the two to each other, the concentration of coupling agents insolution can range from about 0.001% to about 50% weight of thesolution. Preferably the concentration of coupling agents in thesolution ranges from about 0.01% to about 1% weight of the solution, andmore preferably the concentration of coupling agents in the solutionranges from about 0.01% to about 0.1% weight of the solution.

Where a single solution comprises coupling agents and proteins in orderto attach the two to each other, the concentration of proteins insolution can range from about 0.001% to about 90% by weight of solution.Preferably proteins are in solution at a concentration ranging fromabout 0.01% to about 20% by weight of solution, and more preferably theconcentration of proteins in the solution ranges from about 0.01% toabout 10% by weight of solution.

Where proteins in solution are being attached to coupling agents insolution and two solutions are required because the coupling agents andproteins are insoluble in the same solvent, the concentration ofproteins in solution can range from about 0.001% to about 90% by weightof solution. Preferably proteins are in solution at a concentrationranging from about 0.01% to about 20% by weight of solution, and morepreferably the concentration of proteins in the solution ranges fromabout 0.01% to about 10% by weight of solution.

Additionally, where two solutions are required because the couplingagents and proteins are insoluble in the same solvent, the concentrationof coupling agents in solution can range from about 0.0001% to about100% by weight of solution. Preferably coupling agents are in solutionat a concentration ranging from about 0.01% to about 10% by weight ofsolution, and more preferably the concentration of coupling agents inthe solution ranges from about 0.1% to about 10% by weight of solution.

Where a cofactor is required for an enzyme to display catalyticactivity, the cofactor can be presented to a fibrous system via fluid.In an alternative method, the cofactor can be presented to the enzyme byattaching the cofactor to a fiber-forming material by similar methodsdescribed hereinabove regarding the attachment of fiber-formingmaterials and proteins. Where cofactors are attached to thefiber-forming materials of fibers, enzymes can be presented to thecofactors by either attaching them to the fiber or incorporating theminto a fluid that contacts the cofactor.

Where a cofactor is presented via fluid to a fibrous system, theconcentration of cofactors in solution ranges from about 0.0001% toabout 50% weight of solution. Preferably, the concentration of cofactorsin solution ranges from about 0.001% to about 10% by weight of solution.More preferably, the concentration of cofactors in solution ranges fromabout 0.001% to about 10% by weight of solution.

Where a fluid is employed as a means to present an enzyme to a cofactorthat is attached to the fiber-forming material of a fiber, theconcentration of enzymes in solution can range from about 0.001% toabout 50% weight of solution. Preferably, the concentration of enzymesin solution ranges from about 0.01% to about 20% weight of solution.More preferably, the concentration of enzymes in solution ranges fromabout 0.01% to about 10% by weight of solution.

In order to demonstrate the practice of the present invention, thefollowing examples have been prepared and tested. The examples shouldnot, however, be viewed as limiting the scope of the invention. Theclaims will serve to define the invention.

EXAMPLES

Samples of functionalized polystyrene were first synthesized in a mannerwell known in the art. In particular, polymerization of styrene wasconducted in a 20 ml scintillation vial under N₂. In a typicalprocedure, 0.1 g of 2-2′-azobis[2-methyl-N-(2-hydroxyethyl) propionanide(“propionamide”) was first dissolved into 4 ml N,N-dimethylformamide(DMF). The solution was then mixed with 5 ml styrene and 1 ml toluene.The vial was purged with N₂ and incubated in water bath at 72° C. for 24hours. The reaction was stopped via pouring the reaction solution into50 ml of methanol to precipitate polystyrene. The precipitate wasfurther washed by 25 ml methanol for at least 3 times to removeunreacted styrene and initiator. The molecular weight of polystyrene wasmeasured by GPC using a PLgel MIXED column (Polymer Laboratories, MA)with chloroform as the mobile phase.

Polystyrene was then functionalized in a typical manner as follows.Typically, 0.5 g polystyrene and 12.2 mg 4(dimethylamino)-pyridine(DMAP) were dissolved in 8 ml toluene and cooled to 4° C. in a 20 mlscintillation vial. The reaction was initiated by adding 2 ml of 0.01 M4-nitrophenyl chloroformate (NPC) (in anhydrous methylene chloride)under stirring. The reaction was allowed to last 5 hours beforecentrifugation to remove the precipitate of 4-dimethylaminopyridinehydrochloride. The supernatant containing polystyrene attached withnitrophenyl ending groups CPS-NPh) was precipitated and washed withmethanol. The final product was dried by blowing N₂.

Fibers were then made by electrospinning. To that end, a polymersolution was prepared at room temperature by dissolving PS-NPh in amixture of methyl ethyl ketone (MEK) and DMF (v:v=1:1) containing0.5%-wt LiCl. The polymer solution was electrospun following a similarprocedure as reported previously (60) with an electric field strength of0.75 KV/cm. A Teflon capillary tube with an orifice diameter of 0.2 mmwas used as the jet. Fibers were collected on glass slides, stainlessmeshes or aluminum foil for different studies such as SEM analysis andenzyme immobilization. The weight of supporting materials was measuredbefore and after the collection to monitor the net weight of accumulatedfibers.

Enzyme immobilization was accomplished as follows. Nanofibers collectedon stainless mesh were immersed in borate buffer solution (pH 8.2)containing α-chymotrypsin (typically 5 mg/ml). The reaction system wasslightly shaken at room temperature for 36 hours, followed by washingthe fibers with pH 8.2 buffer and deionized water till no absorbance at280 nm was observed in the washing solution. The fibers were finallydried by purging N₂ and stored at 4° C.

The amount of active α-chymotrypsin on nanofibers was determined byactive site titration. Typically, certain amount of nanofibrous enzyme(˜1 mg) was added to 3 ml 4-methylumbelliferyl p-trimethylammoniumcinnamate chloride (MUTMAC) solution (pH 7.5 borate buffer), and theproduct concentration was measured using fluorescence (excitation at 360nm, emission at 450 nm) on a luminescence spectrometer (Model LS50B,Perkin-Elmer Analytical Instruments). Fibers were removed from thesolution by filtration using 0.22 μm PTFE filter beforespectrophotometric measurements.

The hydrolytic activity of α-chymotrypsin was measured usingn-succinyl-ala-ala-pro-phe p-nitroanilide (SAAPPN) as substrate in pH8.2 borate buffer. In a typical measurement for native enzyme, 1 ml of0.8 mM substrate solution was mixed with 10 μl of enzyme solution(0.01˜0.3 mg/ml) in a 1 ml cuvette, and the concentration of thehydrolysis product, p-nitroaniline, was monitored by the absorbance at410 nm. The hydrolysis reaction with nanofibrous enzyme was conducted in20 ml vials. Nanofibers with known weight (˜0.1 mg) were added to 4 mlsubstrate solution under stirring and the absorbance at 410 nm wasmeasured after the removal of the fibers. The time course of thehydrolysis reaction catalyzed by the nanofibrous enzyme was obtainedwith at least 5 reactions started at the same time under the samereaction conditions, but were stopped and analyzed for productconcentration at different times (2˜20 min).

The transesterification activity of α-chymotrypsin in organic solventswas measured at room temperature in hexane or isooctane containingn-acetyl-_(L)-phenylalanine propyl ester (APEE) (concentration rangedfrom 2.5 to 30 mM) and 0.5 M n-propyl alcohol. The solvents receivedfrom the supplier were stored with 3 Å molecular sieves for at least 24h before being used. Typically 5 mg of native α-chymotrypsin or 1 mg ofnanofibrous enzyme was added to 10 ml reaction solution to initiate thereaction. The reaction system was shaken at 200 rpm, and the enzyme wasremoved by filtration using 0.22 μm PTFE syringe filter. The productconcentration was monitored by using Gas Chromatography equipped with aFID detector and a RIX-5 capillary column (0.25 mm×0.25 μm×10 m,Shimadzu). A temperature gradient from 100° C. to 190° C. at a heatingspeed of 20° C./min, followed by 5-min retention at 190° C. was used.The initial reaction rate for the formation ofn-acetyl-_(L)-phenylalanine propyl ester (APPE) was calculated usingdata collected before the conversion reached 5%.

Samples of native and nanofibrous α-chymotrypsin were then incubated inmethanol at room temperature (22° C.). The incubation was stopped atdesired times by purging N₂ to remove methanol. The hydrolysisactivities were then measured using SAAPPN as substrate in aqueousbuffer, according to the procedure described above.

Based upon the foregoing, it will be appreciated that polystyrene with amolecular weight of 200 kDa was synthesized via bulk polymerization,followed by functionalization with nitrophenyl chloroformate. Thefunctionalized polystyrene was then electrospun into nanofibers for useas support materials for enzyme immobilization. It has been reportedthat beaded fibers were often generated in many electrospinningprocesses. The beaded structure, which reduces the surface area/massratio of the fibers, is not desired for enzyme immobilization asconcerned in this work. Initially, spinning of the functionalizedpolystyrene resulted in the formation of beads on the fibers. However,the addition 0.5%-wt of LiCl, which introduces additional charges to thepolymer solution, effectively suppressed the generation of beads. Thediameter of fibers was controlled by varying the concentration ofpolystyrene. Fibers with diameters ranging from 120 nm to ˜1 μm wereprepared and examined for the immobilization of α-chymotrypsin.

The attachment of α-chymotrypsin to polystyrene nanofibers was achievedby immersing the fibers into an aqueous buffer containing the enzyme.The covalent binding was evident in that the enzyme retained on thefiber after extensive wash with buffer and deionized water. The amountof enzyme attached to the fibers was detected via active site titration.For large-scale applications, high enzyme loading is always desired toreduce the size of bioreactors. The enzyme loading on nanofibers isexpected to be high considering the great surface area provided by sucha structure. A theoretical enzyme loading, which varies with thediameter of the nanofibers, can be calculated by assuming monolayercoverage of the outer surface of the fibers. The measured enzyme loadinggenerally increases as the diameter of fibers decreases, following thesame trend as predicted by theoretical calculations. An enzyme loadingof 1.4%-wt was observed with fibers of 120 nm, corresponding to 27.4%monolayer coverage of the fiber.

The stoichiometric enzyme loading of α-chymotrypsin on polystyrene (200kDa) is 11%-wt (assuming 1:1 coupling between the polymer and enzyme),which is higher than the enzyme loading needed for a monolayer coverageof the fibers examined. That implies that it is possible to achieve amonolayer coverage of the nanofibers with the α-chymotrypsin-polystyrenesystem. Several factors may contribute to low enzyme loading. First, thehydrolysis of the functional group, nitrophenyl group, may compete withthe immobilization reaction for active sites on the surface of fibers,and may ultimately lead to the limited attachment of enzyme molecules.This adverse effect can be reduced by increasing the concentration ofenzyme in the reaction solution, and higher enzyme loading was achievedin this way. Second, α-chymotrypsin may undergo autolysis under theimmobilization conditions, and the resulted amino acid fragments mayalso compete for active sites on the fiber. Another imaginable factor isthe availability of functional groups attached to the polymer. It ispossible that certain fraction of the functional ending groups areembedded in the fiber and are not exposed to the outer surface. In thisregard, reducing the diameter of the fibers will increase the exposureof the functional groups and thus improve the enzyme loading.

The hydrolytic activity of the nanofibrous α-chymotrypsin was measuredin aqueous solution, and it accounted 65% of that of nativeα-chymotrypsin for the same hydrolysis reaction. This is a quite highactivity as compared with other forms of immobilization. The apparentactivities of immobilized enzymes are usually much lower than thehomogeneous activities of native enzymes, particularly in the case ofenzymes immobilized into porous materials because of the relativelyhigher diffusional resistance involved there. For example, theeffectiveness factor of α-chymotrypsin immobilized in porous materialswas mostly found in the range of 0.1˜30%. In the case of nanofibrousenzyme, even though the enzyme loading is similar to or even higher thanthose achieved with porous materials, the enzyme is exposed to the outersurface and much less diffusional resistance can be expected. Otherfactors, however, may become predominant in limiting the activity of thenanofibrous α-chymotrypsin. For example, it has been demonstrated thatthe covalent binding of α-chymotrypsin to solid supports led to certainconformational changes of the enzyme molecules, and such conformationalchanges in turn adversely affected the intrinsic reaction kineticparameters of the enzyme. Accordingly, it is believe that the 35%activity loss of the nanofibrous enzyme is mainly caused by thestructural changes due to the chemical attachment of polymers.

In contrast to what observed in aqueous solutions, the apparentactivities of nanofibrous α-chymotrypsin in organic solvents were foundmuch higher than that of native α-chymotrypsin. Nanofibrousα-chymotrypsin exhibited activities that are up to 5670 times higherthan native α-chymotrypsin suspended in the same organic solvents. Theactivity enhancement achieved by the nanofibrous enzyme is even higherthan those observed for α-chymotrypsin solubilized in the organicsolvents via ion-pairing, in which case the highest enhancement was˜2400-fold. This was an unexpected observation because the ion-pairedenzymes provide a homogeneous reaction configuration, which shouldafford the enzyme the upper limiting activity in organic solvents.

It has been well demonstrated that water has a great impact on theactivities of enzymes in organic solvents. For example, the activitiesof alcohol oxidase, mushroom polyphenol oxidase and horse liver alcoholdehydrogenase in organic solvents were found greatly increased upon theaddition of water (up to 10%-v/v). In tests with the nanofibrousα-chymotrypsin of the present invention, the addition of 0.1%-v/v waterto organic solvents also led to a significant increase in the enzymes'activity. However, the increase in native enzyme's activity appeared tobe much more dramatic than nanofibrous enzyme, such that the relativeactivity enhancement (ratio of activity of nanofibrous enzyme to nativeenzyme) decreased noticeably with increase in water content. Oneconsideration is that the added water may not only lead to the hydrationof native enzymes in organic media, but may also improve theirdispersion thus resulted in higher apparent activities. In the case ofnanofibrous enzymes, which have been dispersed well in organic solventsthrough the fibers, the addition of water may only impact enzymeactivity through the hydration mechanism.

Covalent binding of enzymes to solid supports usually improve theenzymes' stability against inactivation induced by structuraldenaturation. Such a stabilization effect was also observed with thenanofibrous α-chymotrypsin. Both native and nanofibrous enzymes werefirst conditioned in anhydrous methanol at room temperature. Methanolwas selected due to its well-known ability to denature proteins. Uponthe removal of methanol by evaporation, the enzymes were reconstitutedinto aqueous solutions and their hydrolytic activities were measured.The half-life time of the nanofibrous enzyme was found to be over18-fold longer than that of the native enzyme.

In another embodiment, Table I presents recipes for three solutions thatwere electrospun to produce fibers. Pure methylethylketone (MEK) wasemployed as the solvent, and the weight percentage of the fiber-formingmaterial, polystyrene nitrophenylchloroformate (PST-NPC), was altered toyield fibers of different sizes.

TABLE I Solution Recipe PST-NPC % by Trial Weight of Solution MEKPST-NPC 1 15 0.5 ml 0.0710 g 2 20 0.5 ml 0.1006 g 3 25 0.5 ml 0.1342 g

PST-NPC can be synthesized using conventional techniques. MEK wasobtained from Sigma Chemicals, Australia.

Enzyme attachment to the electrospun fibers was achieved by immersingthe fibers in a solution comprising alpha chymotrypsin in a boratebuffer having a pH of 8.2. The concentration of alpha chymotrypsin inthe solution was 5 milligrams per milliliter of buffer. The fibers wereimmersed in the solution for approximately 2 days. α-Chymotrypsin wasobtained from Sigma Chemicals, Australia.

Various modifications and alterations that do not depart from the scopeand spirit of this invention will become apparent to those skilled inthe art. This invention is not to be duly limited to the illustrativeembodiments set forth herein.

1. A fibrous protein-immobilization system composition comprising: ananofiber comprising a fiber-forming material; and a protein covalentlyattached to the fiber-forming material, wherein the nanofiber includesat least one functional group suitable to permit the attachment of theprotein, wherein the at least one function group is contained within aportion of the fiber-forming material, and wherein the fiber-formingmaterials are linear polymers selected from the group consisting ofhomopolymers and copolymers of α-olefins, α,β-ethylenically unsaturatedcarboxylic acids, vinyl aromatics, ethyl ethers, and combinationsthereof.
 2. The fibrous protein-immobilization system composition, asset forth in claim 1, wherein the protein is attached indirectly to thefiber-forming material by an inert coupling agent.
 3. The fibrousprotein-immobilization system composition, as set forth in claim 2,wherein the protein includes at least one functional group that canreact with a corresponding functional group on the inert coupling agent.4. The fibrous protein-immobilization system composition, as set forthin claim 1, wherein the protein is a natural or synthetic protein. 5.The fibrous protein-immobilization system composition, as set forth inclaim 4, wherein the protein is selected from the group consisting ofenzymes, hormones, toxins, antibodies, antigens, lectins, structuralproteins, signal proteins, transport proteins, receptors, and bloodfactors.
 6. A fibrous protein-immobilization system compositioncomprising: a nanofiber comprising a fiber-forming material; and aprotein covalently attached to the fiber-forming material, wherein thenanofiber includes at least one functional group suitable to permit theattachment of the protein, wherein the at least one function group iscontained within a portion of the fiber-forming material, wherein theprotein is attached directly to the fiber-forming material, and whereinthe fiber-forming material is selected from the group consisting ofnylons, polyesters, polyurethanes, silanes, or copolymers thereof. 7.The fibrous protein-immobilization system composition, as set forth inclaim 6, wherein the protein includes at least one functional group thatcan react with the at least one functional group on the nanofibercomprising fiber-forming material.
 8. A fibrous protein-immobilizationsystem composition comprising: a nanofiber comprising a fiber-formingmaterial; and a protein covalently attached to the fiber-formingmaterial, wherein the nanofiber includes at least one functional groupsuitable to permit the attachment of the protein, wherein the at leastone function group is contained within a portion of the fiber-formingmaterial, wherein the protein is an enzyme selected from the groupconsisting of chymotrypsin, cytochrome C, trypsin, subtilisin,horseradish peroxidase, soybean peroxidase, and glucose oxidase, andwherein the fiber-forming material is selected from the group consistingof nylons, polyesters, polyurethanes, silanes, or copolymers thereof. 9.A fibrous protein-immobilization system composition comprising: ananofiber comprising a fiber-forming material; and a protein covalentlyattached to the fiber-forming material; wherein the nanofiber includesat least one functional group suitable to permit the attachment of theprotein; wherein the at least one function group is contained within aportion of the fiber-forming material, wherein the protein is containedwithin the fiber-forming material, and wherein the fiber-formingmaterial is selected from the group consisting of nylons, polyesters,polyurethanes, silanes, or copolymers thereof.
 10. A method forsynthesizing a fibrous protein-immobilization system comprising thesteps of: synthesizing a nanofiber comprising a fiber-forming material,wherein the nanofiber includes at least one functional group suitable topermit the attachment of a protein and wherein the at least one functiongroup is contained within a portion of the fiber-forming material; andattaching the protein covalently to the fiber-forming material, andwherein the fiber-forming materials are linear polymers selected fromthe group consisting of homopolymers and copolymers of α-olefins,α,β-ethylenically unsaturated carboxylic acids, vinyl aromatics, ethylethers, and combinations thereof.
 11. The method of claim 10, whereinthe protein is attached to the fiber-forming material after thefiber-forming material is synthesized into a nanofiber.
 12. The methodof claim 10, wherein the step of attaching includes attaching theprotein to a coupling agent and the coupling agent to the fiber-formingmaterial.
 13. A method for synthesizing a fibrous protein-immobilizationsystem comprising the steps of: synthesizing a nanofiber comprising afiber-forming material, wherein the nanofiber includes at least onefunctional group suitable to permit the attachment of a protein, whereinthe at least one function group is contained within a portion of thefiber-forming material, and wherein the fiber-forming materials arelinear polymers selected from the group consisting of homopolymers andcopolymers of α-olefins, α,β-ethylenically unsaturated carboxylic acids,vinyl aromatics, ethyl ethers, and combinations thereof; and attachingthe protein covalently to the fiber-forming material, wherein theprotein is attached to the fiber-forming material before thefiber-forming material is synthesized into a nanofiber.
 14. A method forsynthesizing a fibrous protein-immobilization system comprising thesteps of: synthesizing a nanofiber comprising a fiber-forming material,wherein the nanofiber includes at least one functional group suitable topermit the attachment of a protein, wherein the at least one functiongroup is contained within a portion of the fiber-forming material, andwherein the fiber-forming materials are linear polymers selected fromthe group consisting of homopolymers and copolymers of α-olefins,α,β-ethylenically unsaturated carboxylic acids, vinyl aromatics, ethylethers, and combinations thereof; and attaching the protein covalentlyto the fiber-forming material, wherein the step of synthesizing includeselectrospinning a solution of the fiber-forming material to produce thenanofiber.
 15. A method for synthesizing a fibrousprotein-immobilization system comprising the steps of: synthesizing ananofiber comprising a fiber-forming material, wherein the nanofiberincludes at least one functional group suitable to permit the attachmentof a protein, wherein the at least one function group is containedwithin a portion of the fiber-forming material, and wherein thefiber-forming materials are linear polymers selected from the groupconsisting of homopolymers and copolymers of α-olefins,α,β-ethylenically unsaturated carboxylic acids, vinyl aromatics, ethylethers, and combinations thereof; and attaching the protein covalentlyto the fiber-forming material, wherein the protein is an enzyme and themethod further comprises the step of attaching a cofactor to thefiber-forming material or the step of contacting the enzyme with acofactor in a fluid.
 16. The method of claim 15, wherein the enzyme iscontacted to the cofactor in a fluid.
 17. A method for synthesizing afibrous protein-immobilization system comprising the steps of:synthesizing a nanofiber comprising a fiber-forming material, whereinthe nanofiber includes at least one functional group suitable to permitthe attachment of a protein and wherein the at least one function groupis contained within a portion of the fiber-forming material; andattaching the protein covalently to the fiber-forming material, whereinthe protein is contained within the fiber-forming material, and whereinthe fiber-forming material is selected from the group consisting ofnylons, polyesters, polyurethanes, silanes, or copolymers thereof.